Tetra-substituted benzoquinones and derivatives of the same



Dec. 22, 1964 F. L. CRANE ETAL 3,152,654

worms AND DERIVATIVES OF THE SAME TETRA-SUBSTITUTED BENZOQU Filed Sept.3, 1958 2 Sheets- Sheet 1 WAVE LENGTH Mu 9 7. 4. w OOO OO 450 500 600700 WAVE LENGTH MU INVENTORS. FREDERICK L. CRANE ROBERT L. LESTER DAVIDE. GREEN BY ,4... ma

ATTORNEYS Dec. 22, 1964 F. 1.. CRANE ETAL TETRA-SUBSTITUTEDBENZOQUINONES AND DERIVATIVES OF THE SAME Filed Sept. s, 1958 2Sheets-Sheet 2 WZOEQZ 2 IPOZMJ m N O Q m h m 8 INVENTORS.

FREDERICK CRANE ROBERT L. LESTER DAVID E. GREEN B MI Z'M J 24 7% fak-ATTORNEYS.

United States Patent Ofiice 3,162,654 TETRA-SUBSTHTUTED BENZOQUINGNESAND DERIVATIVES OF TEE SAME Frederick L. Crane, Robert L. Lester, andDavid E. Green, Madison, Wis, assignors to Wisconsin Alumni ResearchFoundation, Madison, Wis, a corporation of Wisconsin Filed Sept. 3,1958, Ser. No. 758,838 Claims. (Cl. 260396) The present inventionrelates to novel products and processes of preparing the same and morespecifically to a new type quinone identified by the term coenzyme Q.The quinone acts as a coenzyme by undergoing cyclic oxidation andreduction during substrate oxidation in mitochondria.

Materials with high respiratory rates generally are good sources fromwhich coenzyme Q can be obtained. The quinone has been obtained, forexample, from beef heart mitochondria, beef liver mitochondria, pigeonbreast muscle cyclophorase, N. crassa, t'orula, Azotobacter, bakersyeast, etc. Saponification of these materials with aqueous alkali metalhydroxide in a solvent such as ethanol in the presence of pyrogallolfollowed by extraction of the saponified mixture with isooctane orhept'ane with or without column chromatography of the extract, is onepreferred general procedure. Pyrogallol is employed as the quinone isirreversibly destroyed in alkaline solutions unless the saponificationmedium contains pyrogallol or equivalent antioxidant. Wherechromatography is employed, e.g. on alumina, magnesium silicate(Forisil), sodium aluminosilicate (Decalso), fullers earth, etc., theuse of isooctane or heptane solutions with elution with a slightly morepolar solvent mixture, e.g. 5 percent ethyl ether isooctane, is also apreferred general procedure. The coenzyme Q preparations also can bepurified by recrystallization from ethanol, methanol, amyl alcohol,ethyl acetate, acetone or acetic acid.

Extraction with heptane or isooctane is preferred over ether, petroleumether, pentane and the like as these latter low boiling solvents extractconsiderably more unwanted or undesirable material. The major impuritieswith heptane and isooctane extraction are the carotenoids and sterolswhich luckily can be removed from the quinone by chromatographictechniques and/or differential crystallization.

Coenzyrne Q preparations derived from different sources under similar ordifferent methods are basically similar in essential properties as notedbelow. All products are yellow-orange, neutral, low melting solids,insoluble in water and soluble in organic solvents including pentane,heptane (n-heptane) isooctane (2,2,4-trimethylpentane), petroleum ether,cyclohexane, benzene, toluene, ethyl ether, acetone, lower alkanols(e.g. methyl, ethyl and amyl alcohols), ethyl acetate, acetic acid,chloroform, carbon tetrachloride and pyridine, with the greatestsolubility being in the non-polar (hydrocarbon) solvents.

The yellow color of the quinone is bleached by reducing agents, e.g. KBHascorbic acid, zinc, etc., and the resulting reduced compound isoxidized slowly in air and regenerated rapidly in the presence of Ag Oto coenzyme Q, i.e. the oxidized form of coenzyme Q. The chromatographicproperties on gel-type precipitated sodium aluminosilicate (availableunder the trademark Decalso) are also similar. Coenzyme Q in isooctanesolution, for example, is adsorbed on Decalso and eluted with diluteethereal (310% ethyl ether) solutions of 3,162,654 Patented Dec. 22,1964 isooctane. All coenzyme Q preparations also reactivate a coenzyme Qdepleted succinoxidase system.

The ultraviolet absorption spectra for coenzyme Q is also qualitativelyidentical throughout the visible region, all preparations in ethanolhaving maxima at about 275 In with a broader band at 405 mu. Thesemaxima disappear upon borohydride (KBH reduction and a single maximum oflower intensity appears at 290 m See FIGURE 1, a and b, where the solidline gives the spectrum for the oxidized form of coenzyme Q and thebroken line the spectrum for the reduced form of coenzyme Q. Spectralevidence also indicates intermediate stages in the reduction. Whencoenzyme Q in ethanol, for example, is shaken withsolid KBH the 275 mpeak disappears and peaks appear at 248 mg and 312 m These latter twobands disappear rapidly followed by the formation of the single stable290 m band. Also, in the visible range when an ethanolic solution ofcoenzyme Q is shaken with small amounts of KBH a pinkish color appearsbefore the yellow color is bleached. The pinkish color appears againupon shaking the bleached solution in air. When this reduction inethanol with KBH, is observed in a recording spectrophotometer a greatincrease in extinction occurs immediately throughout the visible regionwith the transient formation of two sharp bands appearing at 420 and 442m The extinction then drops rapidly until the bleached state is reached.This characteristic is also observed in all coenzyme Q preparationsregardless of source.

When coenzyme Q is hydrogenated at room temperature and atmosphericpressure in a Warburg monometer in ethyl alcohol with palladium oncharcoal as catalyst (23 mg. Pd./n1g. quinone), hydrogen uptake isinstantaneous and complete after several minutes, the values rangingfrom about 1.2 to 1.3 moles H absorbed/ 100 g. quinone. Under similarconditions using platinum oxide in ethanol, a slightly higher value wasobtained.

The infrared spectra of coenzyme Q obtained from various sources bysimilar or different methods is also qualitatively identical. Thisspectra is illustrated in FIGURE II using KBr pellets. As shown here,coenzyme Q exhibits characteristic absorption bands in the infraredregion of the spectrum at the following wave lengths expressed inmicrons: 3.45; 3.52; 4.37; 6.08; 6.20; 6.90; 7.21; 7.77; 7.92; 8.32;8.67; 9.03; 9.78; 10.42; 11.39; 12.55; 13.27.

CoenzymeQ in the reduced form forms an acetate (diacetate) ester whichcan be prepared as follows: 400 mg. of coenzyme Q, obtained from beefheart mitochondria, 400 mg. of zinc dust, 10 ml. of acetic anhydride and2 ml. of triethylamine were warmed for about 510 minutes. The mixturewas transferred to a separatory funnel with ml. of cyclohexane afterfiltering through glass wool. The cyclohexane phase was washed twicewith 25 ml. volumes of 0.3 N HCl and washed three times with 50 ml. ofwater. The solvent was distilled off in vacuo and the semi-solid productwas dissolved with 20 ml. of hot absolute ethanol and allowed to standat 5 C. After two days the practically white precipitate which formedwas filtered by suction and redissolved in 10 ml. of absoluteethanol.After standing overnight at room temperature the diacetate precipitatedand a further small amount was obtained after standing at 5 C. for twodays. After removal of the mother liquor by centrifugation and thedrying of the precipitate in vacuo, the

diacetate derivative of coenzyme Q-lG (see below) was obtained as awhite product which softens at 39 C. and melts at 40 C.

Absorption spectra on the oxidized and reduced forms quinones was thepresence of a considerable number of branched methyl groups as measuredby Kuhn-Roth. Titration of the reduced form of coenzyme Q with analcoholic solution of FeCl -a,ot'-dipyridyl served as the basis ofcoenzyme Q are consistent with the presence of an 5 for determination ofthe equivalent weights reported in cap-unsaturated carbonyl functionundergoing reversible Table III. Using palladium on charcoal as thecatalyst, oxidation and reduction. This indicates that coenzyme Q asnoted above, the moles of H absorbed/mole of cois a quinone and apara-quinone because of the color. A enzyme Q are also reported in TableIII for the various tetra-substituted quinone is also indicated ascoenzyme Q quinones. gives a negative Craven test. Other evidenceincluding TABLE III solubility in lipid solvents also points to thepresence of a quinonoid group or nucleus with a fatty (unsaturatedCmnlyme Q Q- aliphatic) side chain. Permanganate oxidation of coen- Y(using Qi in M KOH) Yields tiriitlir rt ti iit iiiiii 1i? 4? '33? 515ilime acid along with succimc acid and other degradation Moles H /mole11.0 10.2 8.9 1.9 products including partially oxidized products. Thelevulinic acid (identified as the 2,4-dinitrophenylhydrazone) Themolecular Weights and empirical formulas given points t th presence fisoprenoid in Table I best fit the data available although the num- CHSber of carbon atoms assigned to the formulas could be varied shghtlywithout serious conflict with the analytical (R=C CH"CH2OH=R) data. Itis possible that the individual quinone comelements in the side chain ofcoenzyme Q. pounds might also differ slightly in the branching on theThe difierences in melting points and R values (see side chain, in thenumber and position of the double Table I below) as well as noteddifferences in solubility bonds on the side chain as well as involvesome geometriproperties in the lower alkanols (eg. at different temcalisomerism along with various combinations of the peratures) confirmsthat the quinones (coenzyme Q) obsame. It is also possible that thequinonoid nucleus contained from the sources described below aredifierent. tains two side chains with isoprene units instead of onlyPresent evidence shows them to be a homologous series one side chainWhich is isoprenoid in character. of compounds with the same quinonoidchromophore, The following examples will serve to illustrate the inwithtwo alkoxyl substituents and a long side chain which vention. isisoprenoid in character. The compounds appear, at least for the mostpart, to differ only in the length of this Example I' Fmm Beef HeartMnochond' (Q40) chain and specifically in the number of five carbon(isop 1! P 'fi 1057 grams of beef prene) units. This is illustrated inTable I giving propheart mitochondrial protein were worked up batchwiseerties of various coenzyme preparations obtained from 33 in thefollowing manner. One volume of an aqueous susdiiferent sources. pension(-70 mg. protein/ ml.) was added to two vol- TABLE I Source CoenzymeMolecular Empirical Melting Rt Weight Formula Point Value Beet HeartQ,-10 849.3 C53H5304 49.9 (127 T. fltilis (high m. pt. form) Q-9 781. 245. 2 0. 36 A. vimlandi Q,8.. 713.1 37.0 0.42 T. utilir (low 111. pt.form)... Q,7 644.9 30. 5 0.49

Rt values obtained in 8/2 [v./v.] n-ptopanol/HzO with reverse-phasepaper chromatography.

It would be consistent with the data in the above table to assume thatthe alkoxyl groups are methoxy groups and that coenzymes Q-10, Q-9, Q-8and Q-7 have, respectively, 10, 9, 8 and 7 isoprene (C H units. Thecoenzyme Q isolated from bakers yeast is the next lower homologuecontaining 6 isoprene units, i.e. is coenzyme Q-6. It has a meltingpoint of 16 C. and a molecular weight (by FeCl -dipyridyl titration) ofabout 590. The

chemical analysis, given below in Table II, has shown all crystallinecoenzyme preparations to contain no nitrogen, sulfur or halides.

Tests have also shown coenzyme Q to have no vitamin K or vitamin Bactivity and not to restore the prothrombin level of rats which havebeen treated with warfarin.

Methoxyl groups, as noted above, were found to be present in allcoenzyme Q preparations (as judged by the Ziesel determination) and thevalues obtained are reported in Table III. Another constant feature ofall umes of 10 percent KOH in percent ethanol which contained an amountof pyrogallol equivalent in weight to the weight of mitochondrialprotein. This mixture was then refluxed for about 39 minutes.

Step 2: Extracti0n.--T he saponified mixture obtained as above wasextracted three times each with one-sixth volume of heptane. Thecombined heptane extracts were then washed several times with distilledwater until the pH of the aqueous extracts remained unchanged.

Step 3: Evaporation-The washed heptane extract obtained as above wasdried with anhydrous Na SO and the heptane was distilled off in vacuo.

Step 4: Removal of Cholesterol Fraction.--The product or residueobtained as above and containing the desired quinone compoundcontaminated with cholesterol and the like, was dissolved in 250 ml. ofwarm isooctane, and the resulting solution was held at about 5 C. forseveral days. The precipitate which formed was filtered with suction andwashed with 50 ml. of cold isooctane. This precipitate was mainlycholesterol. The filtrate and washings were combined and the isooctancsolution was placed at 20 C. overnight and the small amount ofprecipitate formed was filtered off and discarded.

Step 5: Chromatography.-This purification step was carried out in acolumn containing sodium aluminosilicate (Decalso 50/80 mesh). TheDecalso was first Washed with about 500 ml. of isooctane and the quinoneisooctane concentrate obtained above in step 4 was then added to 5 thecolumn. Elution was carried out as follows collecting approximately 500ml. fractions:

The ether eluate contains the desired quinone and most of it appears infractions 46.

R'echromatography.The ether eluate (e.g. fractions 4, 5 and 6) can berechromatographed in a second Decalso column as above, if desired, asfollows:

Fraction No Solvent Volume Appearance 1 Isooetane .2 '4 5% ethyletherisooctane.

500 Colorless. 1, 500 Single colored baud main color in 2; 4 almostcolorless.

Step 6: Final purificatin.Fractions 2 and 3 of the second column wereconcentrated in vacuo to oil. The oil was heated on a steam bath with100 ml. of absolute ethanol and the hot ethanolic solution containingthe quinone was separated by decantation from a small amount ofinsoluble brownish oil. A yellow precipitate formed inthe ethanolsolution after standing for about a day at C. This precipitate wasfiltered oil and recrystallized from 100 ml. of absolute ethanol afterremoving a very small amount of alcohol insoluble oil. Therecrystallized Q-10 product had a melting point of about 49 C.

Example II.From Beef Heart Mitochondria Without Chromatography (Q10) Thebeef heart mitochondria was saponified with 10 percent alcoholic KOH inthe presence of pyrogallic acid as described above in step 1 of ExampleI. The first heptane extract (see step 2 of Example I) from thesaponified mixture was evaporated and the residue taken up in ethanolleaving a red residue of ethanol insoluble material. The ethanolicsolution containing the desired quinone was held at C. overnight and thewhite crystals that formed (mainly cholesterol) were removed byfiltration and discarded. The orange mother liquor was held at l5 C. forabout 48-72 hours until the quinone crystallizes as orange crystals. Thecrystals are separated by decantation and redissolved in warm 100percent ethanol. The red precipitate which forms at room temperature wasremoved by centrifugation and the ethanolic solution was held at l5 C.overnight. The orange crystals which formed were separated, redissolvedin warm ethanol and again held at l5 C. overnight or until the motherliquor is colorless. The crystals of the desired Q-lO quinone afterseparation from the liquor had a melting point of about 45-47 C.

Example Hl.From Whole Beef Heart Tissue (Q40) Beef heart tissue wastrimmed of fat and connective tissue and passed through a meat grinder.For each pound of heart 600 ml. of 10 percent sodium hydroxide in 95percent ethyl alcohol and 30 gm. of pyrogallic acid were added and themixture refluxed in a water bath set at 90 C. for one-half hour. Theresulting mixture was cooled and placed in a separatory funnel andextracted twice with approximately 100 ml. of heptane. The heptaneextracts were pooled and washed three times with four volumes ofdistilled water. The washed heptane extract was then dried overanhydrous sodium sulfate for about two hours. The heptane was removedunder reduced pressure with gentle heating by a slow stream of steam.The residue which consists of a thin white film overlaid with orange toyellow crystals was taken up in 20 ml. of ethyl alcohol and allowed tostand at 5 C. overnight. After removal of any precipitate that may formon standing, the alcohol was evaporated under reduced pressure with careto avoid extreme spattering in the final stages. The residue was takenup in a minimum volume of isooctane and cooled to l5 C. If any whiteplatelets (cholesterol) form during this cooling step they are filteredoff. The reddish orange isooctane solution was then placed on a columnof sodium aluminosilicate (Decalso 50/80 mesh). (For an extract from 3pounds of beef heart a column of 1%. inches inside diameter by 8 incheslong will suffice.) The column was Washed with isooctane dried oversodium sulfate before addition of the extract.

The column was eluted with dried isooctane until the first yellow band(carotenes) had been completely eluted (about 300 ml. of isooctaneshould be sufficient). The quinone was then eluted from the column with5 percent anhydrous ethyl ether in isooctane (the quinone can beidentified as a yellow band and about 300 ml. of ether solution shouldbe sufficient). Cholesterol and other impurities may appear in theeluate in the last yellow fractions or immediately thereafter, so thefinal tailing fractions of the quinone should not be mixed with the bulkof the material from the main portion of the yellow band. The yelloweluates which contain the main portion of the yellow quinone band werepooled and the solvent evaporated. The yellow oil remaining was taken upin a minimum volume of hot ethanol and allowed to stand at 5 C.overnight. The orange crystals which form on standing were removed byfiltration and washed with cold (5 C.) ethanol. The crystals of thedesired Ql0 quinone can be further purified if desired, byrechromatographing on Decalso or recrystallizing from ethanol or both.Additional material can also be recovered by reprocessing the motherliquor through a second chromatographic column in accordance with theprocedure described above.

Example I V.-F rom Azotobacter vinelandii (Q-8) This process was carriedout in accordance with Example I except for the omission of step 4 andincludes saponification, extraction, evaporation and chromatogexcept forthe difference in melting point and for the fact that the Azotobacterquinone is more soluble in methanol, ethanol and glacial acetic acid,also appear to be one and the same.

Example V.Fr0m Torula (Q-7 and Q-9) Step 1: Saponificatiom-The followingmixture was refluxed for 30 minutes: 1 liter 15 percent w./v. KOH inpercent ethanol, 37 g. pyrogallol and 330 g. of dried Torula powder(feed grade) slurried in 800 ml. water. Eight batches were refluxed inthis manner.

Step 2: Extraction-Each batch was extracted 3 times with 400 ml. volumesof isooctane in a separatory funnel. The last extract of one batch wasused to extract a fresh batch. The pooled isooctane extracts were driedover anhydrous Na SO4, and evaporated in vacuo to a volume of 500 ml.After standing overnight at -20 C., a precipitate formed and wasdiscarded after ccntrifugation. The supernatant solution contained 8.15g. of impure coenzyme Q, i.e. a mixture of Q9 and Q-7.

Step 3: Silicic acid chromatography.The material from step 2 wasadsorbed on a column containing 300 g. of silicic acid and g. ofdiatomaceous silica (Super- Cel) equilibrated with isooctane. Thematerial was eluted with l/l (v./v.) chloroform/isooctane. The fractionscontaining coenzyme Q, which emerged as a deep orange band, were pooledand evaporated to dryness in vacuo.

Chromatography on Decalso columns also has been used successfully forthis step, essentially as described in Example I.

Step 4: Separation of Q-9 and Q7 by reversed phase paperchrmat0graphy.The residue from the previous step was dissolved in 50 ml.of ethanol. This ethanolic solution was applied (1 mg. coenzyme Q/cm.)to Whatman No. 17 filter paper 4 cm. from the bottom. The paper had beenpreviously treated with silicone (Dow Corning No. 550). The paper wasthen hung in the chromatography chamber and allowed to equilibrate with7/3 (v./v.) n-propanol/H O for about 24 hours. After the equilibrationperiod the material was chromatographed in the ascending manner with theabove-mentioned solvent system. When the solvent front had progressed 20cm. or more two distinct yellow bands could be observed. The papers werethen air dried and the appropriate bands were cut out. The upper andlower bands contained the low melting (Q-7) and high melting (Q9)compounds, respectively. The quinones were separately eluted from thepapers with warm ethanol until no color remained. The alcohol eluateswere filtered to remove paper particles and then evaporated to dryness.The small amount of silicone which was now present was removed bysilicic acid chromatography essentially as described in step 3 exceptthat smaller columns were now used. From these columns were obtained thefollowing: high melting Q-9 compound, 80 mg.; low melting Q-7 compound,465 mg.

Step Final purification. (a) High melting comp0und.-The Q-9 eluate fromthe silicic acid column was taken to dryness and the residue wasdissolved in 10 ml. absolute ethanol. After a day at 5 C., a smallamount of whitish precipitate had formed and was discarded aftercentrifugation. The supernatant was placed at C. overnight. The yellowprecipitate which had formed was centrifuged off and redissolved in 8ml. of warm 1/1 (v./v.) methanol-ethanol and placed at 5 C. The yellowcrystals which formed were recrystallized from 4 ml. of ethanol-methanolsolution at room temperature. Yield: 44 mg. of Q-9, M.P. 43 to 45 C.More compound can be obtained from the mother liquors.

(b) Low melting f0rm.--The Q-7 eluate from the silicic acid column wastaken to dryness, the residue taken up in 36 ml. 4/1 (v./v.)ethanol/methanol and placed at 5 C. overnight. A small amount ofcolorless oil separated and was discarded after centrifugation. Thesupernatant was placed at 20 C. overnight. The yellow precipitate whichformed was centrifuged and redissolved in 12 ml. 4/ 1 ethanol/methanoland placed at 5 C. for two days. The crystals were filtered off in thecold and recrystallized from 7 ml. of the ethanol-methanol solventyielding 233 mg. of Q-7 crystals, M.P. 30 C. More material can berecovered from the various mother liquors.

Example V1.-From Beef Heart Mitochondria (Q-JO) Direct extraction.-Beefheart mitochondria in a suspension of about an equal volume of water wasextracted with ten volumes of ethanol overnight at room temperature. Theethanol extract was then cooled and held at 15 C. overnight and theprecipitate which formed was separated by filtration. The solvent wasthen evaporated from the extract and the residue was taken up in aminimum volume of petroleum ether (boiling point 3060 C.). The petroleumether extract was poured slowly into ten volumes of acetone and theprecipitate removed by filtration. The acetone was evaporated and theacetone solubles were taken up in a minimum volume of heptane and placedon an acid washed chromatographic alumina column. The column was elutedwith heptane .mum volume of warm acetic acid. On standing overnight at 5C. orange crystals of Q-l0 formed which after recrystallizing fromacetic acid had a melting point of about 50 C.

Example VII.Fr0m Torula utilis (Q-9 and Q-7) Direct extracti0n.The drytorula yeast was stirred intermittently with isooctane for about l-2days and then the yeast was allowed to settle and the yellow supernatantwas siphoned ofi. Sufficient isooctane was used to provide a supernatantequivalent to about one-fifth the total volume of the suspension. Theisooctane supernatant was placed on a Decalso (sodium aluminosilicate)column until the quinone started passing through when a new column wasstarted. The quinone was eluted from each column with 5 percent ethylether in isooctane and the eluant concentrated to an orange oil.Chromatography using fullers earth and the like can also be employedwith further purification by crystallization from ethanol and the likeas described above.

The coenzyme Q products obtained from torula upon fractionalcrystallization in methanol or ethanol, yield two closely relatedproducts as described above. Although one product (Q-9) was found tohave a melting point after several recrystallizations of about 45.2 C.and the other product (Q-7) a melting point of about 30.5, both productshad the same visible spectra and the same ultraviolet spectracharacteristic of all coenzyme Q products. Coenzyme Q6 can be obtainedfrom the nonsaponifiable fraction of bakers yeast in accordance withExample V.

In the above examples, Examples I-V employ saponification and ExamplesVI and VII direct extraction. There is considerable evidence thatcoenzyme Q obtained by direct solvent extraction followed bypurification through chromatography and crystallization is moreefficient enzymatically than coenzyme Q isolated by a procedureinvolving saponification. Coenzyme Q-10 obtained from beef heart bydirect extraction, for example, can be fully reduced in the presence ofcatalytic amounts of mitochondria, cyanide and succinate, Whereas only apart of coenzyme Q-10 obtained from beef heart by saponification can bereduced enzymatically even though it is completely reducible chemically.

Coenzyme Q products are mild anti-coagulants, i.e. have slightanti-vitamin K activity. Extraction of coenzyme Q from mitochondriadecreases succinoxidase activity and addition of coenzyme Q restores theinitial activity. Crane et al., Biochim. Biophys. Acta., 25, 220 (1957).In view of this coenzyme Q can be used in assaying for succinic acid orthe succinate radical and is a valuable laboratory tool for assays inthis field. This can be readily done by extracting mitochondria withisooctane, adding coenzyme Q to the extracted mitochondria (which is nowspecific for the succinic acid or succinate radical) and then adding thesample to be tested. By measuring the oxygen uptake the amount as wellas the presence of succinic acid or the succinate radical in the samplecan be readily obtained. Coenzyme Q, as pointed out above, undergoesreversible oxidation and reduction and while derived from naturalproducts neither the oxidized form nor the reduced form are known innature. In this connection, it has been demonstrated that whilecrystalline coenzyme Q isolated in accordance with the examples is notwater-soluble and has an ultra-violet absorption maxima at about 405 mthat the coenzyme Q active factor as it occurs in lipoprotein in natureis water soluble and has the corresponding maxima at about R=H, Acyl,etc., n=6-10. 425 mp. It has also been demonstrated that the coenzyme Qactive factor as it occurs in lipoprotein in l nature (apparently as alipoprotein-coenzyme Q com- CH3O plex) is about five times as efficientas the crystalline co- 5 H3 enzyme Q products of the present inventionin the restora- OHaO (CH2OH2 (LHCH,) nH tion of succinoxidase activityin isooctane extracted mitochondria. This figure of five times aseflicient was also 6 obtained by assaying in the presence ofphospholipid al- IV though it 18 known that some phospholipid materialfrom 10 10 the lipoprotein facilitates the utilization of coenzyme Q i bf 1 F 1 I by isooctane-extracted mitochondria. e a (We ormu 0mmrepresents Investigations to date have established that cocnzyme 3 assoimnmes refined 2, i i m Its Q may be represented by the followingbasic formula: lze or qumone and olzmu =H) mine sents coenzyme Q in itshydroquinone form or, as some- 0 times referred to, coenzyme Q in itsreduced form. In both Formulas I and H the hydrocarbon side chain OHremains unsaturated. In Formulas III and IV the side a 3 (:11 chain issaturated and Formula III (R=H) represents 5 3 h dr ted Q 't h d C u yogena coenzyme 1n 1 s y roqumone or re- CHQO H2011 CH2) H duced form,and Formula IV represents hydrogenated coenzyme Q in its quinone oroxidized form.

I Coenzyme Q, as pointed out above, is thus characterized by a quinonoidnucleus which is further charactered by having, as substituents, twomethoxy groups and Where For for The meth l rou alon with un atu ated hdrocarbon hydmquinone form of coenzyme and its filenvafives :hain 0 hfhe guinong ring. The uns aturateti hydrocarmay be represented by thefollowmg formula" bon side chain, as shown by the data, can vary inlength, OR and is characterized by the presence of a multiple ofisoprene or like unsaturated groups. As far as known,

CH3 this structure, i.e. the structure of coenzyme Q, repres sents abasically new type of substituted quinone. De-

rivatives can be made as shown by the fact that the hydroxyl groups ofthe reduced or hydroquinone form are e reactive groups and can bereadily acylated, e.g. con- H verted to lower alkanoyl derivatives suchas the diacetate described above. The side chain is also reactive asshown Whe and WL c. The hydroquiby its hydrogenation described above,and in addition to 110116 can be P P With 4 reduction in the resultingsaturated side chain, other derivatives can be t a l as described above,and the preparation of y made in accordance with established chemistryinvolvderivatives (R=acyl) from the hydroquinone (C y ing the reactionof various reactants with unsaturated Q in reduced form) can be preparedas described above hydrocarbons,

in connection with the preparation of the diacetate ester. The presentapplication is a continuation-in-part of Hydrogenated coenzyme Q withthe i e chain our application Serial No. 746,787, filed July 7, 1958,saturated) can be prepared in the hydroquinone form now b d d withhydrogenation over a hydrogenation catalyst as de- W l i scribed above.Hydrogenated coenzyme Q can also be 1 A d represented by h f ul preparedin the quinone form by the following process. 0R

Hydrogenation of coenzyme Q over Raney nickel catalyst in ether solutionor over platinum catalyst in OH3O CH3 dioxane solution was carried outat 1 atmosphere pres- O sure. Eleven molecular equivalents of hydrogenwere absorbed giving the hydroquinone form of hydrogenated SE30(OHZCHPHGHDnH coenzyme Q of Formula III below (R=H, 11:10). This 6compound was obtained as a colorless oil by filtration to remove thecatalyst then evaporation to remove the sol- 5 where and R 15 Selected mthe group consistvent. The hydroquinone may be acetylated by treatmg ofhydrogen and lower alkanoylgroups' ment with a small excess of aceticanhydride. (Formula A Product represented by the formula: III, R= acyl.)o

If the oily hydrogenation product is oxidized by treatl ing it (in ethersolution) with silver oxide, the eicosa- O a 3 hydrocoenzyme of Formula1V below (n=l0) is formed, E

omoonion,- HOHmH M523? 278 m EiZ 187. H o Analysis.-Calcd. for C H O C,80.19; H, 12.55. Found: c, 80.30; H, 12.36. f yp process of preparing atetra-substituted benzo- QR quinone of the formula: R CHaO- CH3 onao- GHCH O- cr cn ononnsn CH3 CH3() (CH OH=(!3-CHz)nH R III O where n=6-10,which comprises saponifying materials with high respiratory ratesselected from the group con sisting of beef heart tissue including beefheart mitochondria, Azotobacter vinelandii, Torula and bakers yeast, inan aqueous ethanolic reaction mixture containing alkali metal hydroxideand pyrogallol, extracting the saponified mixture with a solventselected from the group consisting of heptane and isooctane andrecovering the tetra-substituted benzoquinone in the resulting extract4. The process of claim 3 Where the tetra-substituted benzoquinone ischromatographed in isooctane solution and eluted with an etherealsolution of isooctane.

5. The process which comprises the direct extraction 12 of the materialsof claim 3 with ethanol and in Which the resulting tetra-substitutedbenzoquinone is chromatographed as in claim 4.

References Cited in the file of this patent Crane et al.: Biochem etBiophy. Acta, v01. 25, July 1957 (pages 220-221).

Crane et al.: Biochem et Biophy. Acta, vol. 22, 1956, pages 475-485.

Green et al.: J. Biol. Chem., vol. 217, pages 551-561 1955).

Vischer: J. Chem. Soc., 1953, pages 815-820.

1. A PRODUCT REPRESENTED BY THE FORMULA:
 2. A PRODUCT REPRESENTED BY THEFORMULA:
 3. A PROCESS OF PREPARING A TETRA-SUBSTITUTED BENZOQUINONE OFTHE FORMULA: